14 October 2009. Dimebon appears as one bright light in a field of less-than-stellar clinical trial results of late. But like many stars that suddenly explode into view, is the compound destined to become a black hole? While the drug appeared to stave off clinical decline in mild to moderate Alzheimer patients in a Phase 2 trial, and even manage some improvement (see ARF related news story), no one knows exactly how it works. Its established binding to acetylcholinesterase is too weak to explain cognitive effects, and though there are indications Dimebon reduces mitochondrial vulnerability to stress, it is not clear how it does this or whether it has any clinical significance.
According to drug sponsor Medivation, Inc., the small molecule, originally marketed as an antihistamine, inhibits brain cell death in preclinical models, but again there is no evidence that this is how it affects cognition in humans. There is also a growing list of receptors for which the drug has high affinity, suggesting that it may work as a neurotransmitter enhancer, after all. And topping off the uncertainty, evidence presented at the International Conference on Alzheimer’s Disease (ICAD), held last July in Vienna, Austria, suggests that Dimebon enhances the release of amyloid-β (Aβ) in three different cell and animal models of Alzheimer’s.
The media pounced on this surprising news (see Forbes.com and Reuters). Scientists, too, scratched their heads, wondering what the results mean for the amyloid hypothesis of AD, where most drug developers, including Medivation’s pharma partner Pfizer, mostly try to decrease Aβ with their experimental medicines. At the same time, some researchers have privately questioned the quality of some of the mechanistic research Medivation researchers have been presenting. All told, there seem to be more questions about this drug than answers, and only time and further efficacy trials will tell whether it fizzles or continues to shine. “What we have are inchoate observations that are perfectly fine and perfectly interesting to talk about, but they have no bearing on what Dimebon does or doesn’t do in an Alzheimer’s brain,” said Rachelle Doody, Baylor College of Medicine, Houston, Texas, in an interview with ARF. “The one thing we know so far is that our preliminary signal is that it’s efficacious.” Doody was the senior investigator on the recent clinical trial of Dimebon in AD (see ARF related news story).
Dimebon and Aβ
Sam Gandy, Mount Sinai School of Medicine, New York, revealed the Aβ connection at ICAD. Gandy collaborated with John Cirrito, University of Washington School of Medicine, St. Louis, Missouri, to investigate the effects of Dimebon on Aβ production in cellular and animal models. Cirrito has pioneered a microdialysis assay that measures levels of Aβ in brain interstitial fluid (ISF), and he previously showed that synaptic activity increases ISF Aβ in mice (see ARF related news story). With WashU colleague David Holtzman, Cirrito recently reported that ISF Aβ falls and rises with the sleep/wake cycle (see ARF related news story), and another recent study tied ISF Aβ levels to brain activity in recovering trauma patients (see ARF related news story). The interpretation is that ISF Aβ may reflect synaptic activity.
In Vienna, Gandy showed that acute (six-hour) treatment with even picomolar levels of Dimebon increased release of Aβ from N2A neuroblastoma cells overexpressing human APP with the Swedish mutation. The largest effect, an almost twofold increase, occurred at a concentration of 50 nM Dimebon, which is similar to the concentrations Medivation claims protect mitochondria and promote neurite outgrowth. At micromolar concentrations Dimebon also increased release of Aβ from isolated synaptoneurosomes, a model for synaptic vesicle release. Within three minutes of drug treatment, this preparation released 10 percent more Aβ40/42 than controls, but by five minutes after treatment, Aβ release was back to normal. These two in-vitro observations got backup from an in-vivo study in Tg2576 transgenic mice, where a single intraperitoneal injection of the drug led to an increase of Aβ40 in the ISF at nine hours post-treatment. There was no statistically significant Aβ elevation prior to the nine-hour timepoint, and by 10 hours, levels were back to normal.
How these observations relate to Dimebon action in Alzheimer patients is anyone’s guess. “The available data tell us very little at the moment,” Cirrito said. “Acutely, Dimebon can increase Aβ levels outside the cells, but what that means for Alzheimer disease is still unknown, because people are not taking the drug for 10 hours,” he said. Doody agreed. The cell culture experiment suggests the drug may increase release of Aβ, but it is hard to draw firm conclusions, she suggested. “Remember, these are abnormal Aβ-processing cells. Dimebon did something that resulted in an increase of total amyloid, possibly helped them get rid of it, which would be beneficial,” she told ARF. She also questioned the value of the synaptoneurosome experiments. “What this is a model of I’m not sure, maybe synaptic activity, but they are not hooked up to anything, there’s no electrophysiology, and it gets a transient small increase in Aβ40/42. I can draw no specific conclusions from that.” Ilya Bezprozvanny, University of Texas Southwestern Medical Center, Dallas, voiced similar sentiments in an interview with ARF. “It seems to me that Aβ increases, even in cells expressing APP-Swedish, suggest the [Dimebon] effect does not depend on synaptic activity,” he told ARF. Bezprozvanny was not involved in the Aβ experiments, but his group has evaluated the effect of Dimebon in cellular models of Huntington’s, another disease Dimebon is being tested on in Phase 3 (see below).
Those who doubt the amyloid hypothesis of AD might see vindication of sorts in a drug that seems to slow the progression of cognitive decline in AD while increasing brain Aβ. But the two may not necessarily be mutually exclusive. Gandy suggested in Vienna that Dimebon may be protective because it helps remove Aβ from inside neurons, where it may do most the damage. Andy Protter, of Medivation, does not see the Aβ effect as an issue for Dimebon. “The ambiguity in what it means certainly fits with ambiguity of what Aβ does in the brain,” he said. “There’s a lot of confusion about what we expect drugs to do and how we want them to do it.” For her part, Doody sees no relation between Dimebon’s acute effects on Aβ release and the Aβ hypothesis. “In no way does this negate the Aβ hypothesis, and it does not negate Dimebon,” she said.
Mechanism of Action
Exactly what Dimebon does is a bit contentious. So far there are several theories. Medivation claims that Dimebon protects both mitochondria and neurons. At the 9th International Conference AD/PD, held this past March in Prague, Czech Republic, Maria Ankarcrona from the Karolinska Institute, Stockholm, Sweden, presented data to suggest that nanomolar concentrations of the drug increase the mitochondrial membrane potential in primary cortical neurons from mice and also improve mitochondrial resistance to ionomycin. “Our impression is that Dimebon has some kind of enhancement of mitochondrial function and strengthens the mitochondria in some way to resist stress,” she told ARF. But she admits there is no link yet between mitochondrial membrane potential increases and synaptic function or cognition. “We would like to make that link, but we are not at that stage yet,” she said. She also noted that the drug could have functions other than mitochondrial.
Protter and colleagues at Medivation have similar mitochondrial data, presented at the December 2008 American College of Neuropsychopharmacology meeting in Scottsdale, Arizona, suggesting improved resistance to ionomycin at nanomolar concentrations. They also reported that Dimebon increased migration of mitochondria to neurites of hippocampal neurons and stimulated neurite outgrowth at concentrations as low as 0.1 nM. At higher concentrations (100 nM) it appears to protect mitochondria from the toxic effects of Aβ42.
How Dimebon targets mitochondria is unclear. One paper reports that it can bind to mitochondrial transporters and the mitochondrial permeability transition pore (see Bachurin et al., 2003), which may be one mediator of the toxic effects of Aβ (see ARF related news story). Another possibility is that the drug somehow prevents aggregation of Aβ, since recent evidence suggests it can reduce aggregation of TDP-43, another amyloidogenic protein (see Yamashita et al., 2009).
Bezprozvanny voiced skepticism about the mitochondrial permeability transition pore theory. Though he was unable to obtain the compound from Medivation for testing, Nanosyn Inc., a contract research organization, synthesized it and Bezprozvanny used that preparation in cell toxicity assays. He found that micromolar, not nanomolar, concentrations were required to protect from glutamate toxicity in a cell model of Huntington disease (medium spiny neurons expressing an expanded huntingtin fragment on a yeast artificial chromosome; see Wu et al., 2008). Because both glutamate and ionomycin cause an influx of calcium that leads to mitochondrial stress, Bezprozvanny doubts that Dimebon is protecting mitochondria. “If Dimebon protects mitochondria against ionomycin, then we should have seen protection in glutamate toxicity assays in the nanomolar concentration range,” he told ARF. Instead, he only saw mitochondrial protection effects at concentrations 10 μM and above, where Dimebon blocks NMDA receptors. Bezprozvanny also questioned the relevance of ionomycin assays. “Ionomycin is completely artificial, whereas glutamate is a physiological stimulus that mimics excitotoxicity,” he told ARF. People don’t like the ionomycin experiments because it is non-specific and a very severe toxin, suggested Doody, “but you have to start somewhere.” She said that there is now a larger proprietary program underway to understand Dimebon’s mechanism.
In fact, the strongest link between Dimebon and cognition may be neuroreceptors. An early Russian study showed that the drug inhibits acetylcholinesterases, NMDA-type glutamate receptors, and L-type calcium channels (see Lermontova et al., 2001), but the concentrations needed are much higher than what is needed for cognitive protection, according to Protter (who claimed that Medivation has a paper in review on the subject). Work from Bezprozvanny’s lab supports this. It showed that micromolar concentrations are needed to block NMDA receptors and voltage-gated calcium channels in striatal neurons from wild-type or Huntington disease mice expressing a polyglutamine expanded huntingtin. “It is hard to believe Dimebon has physiological effects through these targets,” Bezprozvanny told ARF. On the other hand, Doody feels that mitochondria are where it’s at. “From what’s in the public domain, I personally believe the most convincing hypothesis is that Dimebon is working at picomolar concentrations by protecting mitochondria. Exactly how it is doing that has not been either discovered or disclosed,” she told ARF.
There are some alternatives to the mitochondria hypothesis. Bezprozvanny’s lab has found a number of other receptors to which Dimebon binds with high affinity. These include: α1B, α1D, and α2A adrenergic receptors; H1, H2, and Imidazoline I2 histamine receptors; and 5-HT2c, 5-HT5A, and 5-HT6 serotonin receptors (see Wu et al., 2008). It is unclear how these receptors might fit with the cognitive effects of Dimebon, “but you can imagine that all the cognitive effects are due to effects on central receptors,” he said. In support of this idea, Michael Marino and colleagues at Cephalon Inc., West Chester, Pennsylvania, report in the upcoming October 15 Biochemical Pharmacology that Dimebon binds to 5-HT6 receptors and has acute cognition-enhancing activity (see Schaffhauser et al., 2009). First author Herve Schaffhauser and colleagues show that Dimebon inhibits the receptor with moderate affinity in vitro (Ki is 25 nM) and occupies the receptor in vivo with similar kinetics to the selective 5-HT6 antagonist SB-399885, which has been touted as a potential treatment for AD and schizophrenia (see Hirst et al., 2006). Schaffhauser and colleagues show that Dimebon improves short-term memory in a social recognition model; that is, adult rats given the drug were better able to remember a juvenile rat placed in the same cage.
Currently, there are two 5-HT6 antagonists in clinical development for AD. GlaxoSmithKline has completed two Phase 2 trials to study the effects of SB-742457 (see ClinicalTrials.gov’s NCT00348192 and NCT00224497) and is currently recruiting for two more (see NCT00708552 and NCT00710684). Suven Pharmaceuticals of Hyderabad, India, has completed a Phase 1 trial of its 5-HT6 antagonist SUVN-502 (see ICAD abstract). Doody agreed that the 5-HT6 activity could be relevant to the mechanism of action of Dimebon but thinks it may be but part of the picture. “It looks like the drug might have both symptom-improving and disease-modifying action. No one is claiming to have fully elucidated this, but it is being carefully studied,” she said.
At the end of the day, the crucial test for Dimebon will be whether the clinical data hold up. There are several Phase 3 trials currently underway for patients with mild to moderate and moderate to severe AD and Huntington disease, including several multinational trials and trials of patients already taking memantine or memantine and donepezil (see ClinicalTrials.gov).—Tom Fagan.